A major area of interest in genome biology, especially in light of the determination of the complete nucleotide sequences of a number of genomes, is targeted integration into genomic sequences. Attempts have been made to alter genomic sequences in cultured cells by taking advantage of the natural phenomenon of homologous recombination. See, for example, Capecchi (1989) Science 244:1288-1292; U.S. Pat. Nos. 6,528,313 and 6,528,314.
In addition, various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination and targeted integration at a predetermined chromosomal locus. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. For example, targeted integration using zinc finger nucleases has been demonstrated with circular (plasmid) DNAs having long (˜750 base pair) homology arms. See, Moehle et al. (2007)Proc. Nat'l. Acad. Sci. USA 104(9):3055-3060.
Targeted integration relies on manipulating normal cellular processes. Typically, cells often depend on homology-directed repair (HDR) which heals spontaneous double strand breaks (DSB) in the genome using the sister chromatid as a template. For targeted insertion of exogenous sequences of interest, the exogenous DNA sequence is constructed so that it is between the two regions in the donor plasmid that contain homology to the genomic location being targeted. The cellular DNA repair machinery will unwittingly copy this genetic information into the chromosome while healing any spontaneous DSB that may have occurred (See Thomas et al, (1986) Cell 44:419-428 and Koller et al, (1989) Proc Natl Acad Sci USA 86: 8927-8931).
In plants, biotechnology has emerged as an essential tool in efforts to meet the challenge of increasing global demand for food production. Conventional approaches to improving agricultural productivity, e.g. enhanced yield or engineered pest resistance, rely on either mutation breeding or introduction of novel genes into the genomes of crop species by transformation. Both processes are inherently nonspecific and relatively inefficient. For example, conventional plant transformation methods deliver exogenous DNA that integrates into the genome at random locations. Thus, in order to identify and isolate transgenic lines with desirable attributes, it is necessary to generate thousands of unique random-integration events and subsequently screen for the desired individuals. As a result, conventional plant trait engineering is a laborious, time-consuming, and unpredictable undertaking. Furthermore the random nature of these integrations makes it difficult to predict whether pleiotropic effects due to unintended genome disruption have occurred. As a result, the generation, isolation and characterization of plant lines with engineered genes or traits has been an extremely labor and cost-intensive process with a low probability of success.
Targeted gene modification overcomes the logistical challenges of conventional practices in plant systems, and as such has been a long-standing but elusive goal in both basic plant biology research and agricultural biotechnology. However, with the exception of “gene targeting” via positive-negative drug selection in rice or the use of pre-engineered restriction sites, targeted genome modification in all plant species, both model and crop, has until recently proven very difficult. Terada et al. (2002) Nat Biotechnol 20(10):1030; Terada et al. (2007) Plant Physiol 144(2):846; D'Halluin et al. (2008) Plant Biotechnology J. 6(1):93.
Creation of a targeted DSB can dramatically increase the frequency and specificity of transgene integration (Rouet, P., et al (1994) Proc Natl Acad Sci USA, 91: 6064-6068). The custom engineering of site-specific nucleases has therefore accelerated targeted integration technology. Zinc-finger nucleases (ZFNs) are fusions between zinc-finger DNA binding domains and the nuclease domain of the type IIs restriction enzyme FokI. When two such ZFN fusions bind at adjacent sites on the chromosome, the nuclease domains interact to create a double-strand break in the DNA. The non-homologous end-joining (NHEJ) pathway can directly ligate the broken ends together, often with a gain or loss of several base pairs (Weterings and van Gent (2004) DNA Repair (Amst), 3:1425-1435).
Previous investigators have found that non-specific DNA can be captured at a site of double-strand break repair in S. cerevisiae (Havi-Chesnner et al, (2007) Nucleic Acids Res, 35, 5192-5202). In addition, repetitive element and mitochondrial DNA fragments have also been observed to integrate at the site of DSBs in S. cerevisiae (Moore, J. K. and Haber, J. E. (1996) Nature, 383, 644-646 and Yu, X. and Gabriel, A. (1999) Mol Cell, 4, 873-881). It is known that while many organisms, including mammals, plants and filamentous fungi tend to rely mainly on NHEJ for healing DSBs, S. cerevisiae is considerably more likely to use HDR for healing these lesions (Ishibashi et al, (2006) Proc. Natl. Acad. Sci. USA 103(40): 14871-14876). Thus it is very difficult to draw conclusions about DSB repair mechanisms in mammalian cells based on experiments performed in S. cerevisiae. Exogenous single-stranded oligonucleotides have been used to repair DSBs in yeast via single strand annealing (SSA) but this homology-based repair process is fundamentally different from NHEJ repair (Storici et al. (2003) Proc Natl Acad Sci USA, 100:14994-14999, Storici et al. (2006) Mol Cell Biol, 26:7645-7657).
In murine fibroblast cells, researchers were able to induce a DSB using the homing endonuclease Sce-I and found that exogenously introduced fragments from nuclease-digested φX174 genomic DNA could be captured in the break. Sequencing of the junctions revealed regions of microhomology between the two DNAs (Lin, Y. and Waldman, A. S. (2001) Nucleic Acids Res, 29, 3975-3981). Other experiments revealed the capture of other exogenous fragments of DNA (Lin, Y. and Waldman, A. S. (2001) Genetics, 158, 1665-1674). However, this result has limited practical applicability in that the researcher is bound to induction of a DSB by the Sce-I homing endonuclease and thus the location of interest must either contain a Sce-I site naturally, or the researcher must go through the arduous process of inserting a Sce-I site through random integration or some other such technique. Alternate techniques for exogenous DNA introduction rely on the practioner having extensive knowledge about the sequence identity of the region for these techniques often depend on fortuitous DSBs occurring within large stretches of homology (six or seven kilobases of DNA) between the donor and the genomic region being targeted. Recently, shorter regions of homology (50-100 bp) have been demonstrated to be functional in HDR when coupled with creation of a targeted double strand break (see co-owned U.S. Patent Publication No. 20090263900).
Therefore, to date, donor molecules have not been shown to be integrated directly into the site of cleavage. Thus, there remains a need for additional methods and compositions to allow targeted insertion of desired donor nucleic acids in cells to produce a precise, investigator-specified allele at an endogenous locus.